For in vitro studies, cSCC cells were treated as indicated of each individual experiment. Cells were rinsed with PBS and lysed in buffer containing Triton X-100 (1%), TrisHCL (0.05M), NaCl (0.1M) and protease/phosphatase inhibitors and EDTA (5mM). Lysates were centrifuged to extract protein at 15,000 rpm. Protein was quantified using Bradford analysis followed by denaturing with sample buffer containing a reducing agent. Proteins were separated on a 4–12% gradient gel, transferred on PVDF membrane, blocked in Non-fat milk (5%) and incubated in primary antibodies overnight at 4 degrees. Blots were rinsed, incubated with the 2nd antibody and developed using ECL western blotting solution (BioRad). Protein levels of AKT, pAKT, S6, pS6, p70S6K, pp70S6K were all determined by Western blotting using antibodies from Cell Signaling Technology (Danvers, MA). Levels of FGFR2 was detected using anti-FGFR2 (sc-53219, Santa Cruz Biotechnology, Santa Cruz, CA) antibody. Primary antibody against β-actin was purchased from Abcam (Cambridge, MA).
Primary antibody against β actin
Manufactured by Abcam
Sourced in United Kingdom
Primary antibody against β-actin. This antibody is used to detect the β-actin protein, which is a widely-expressed cytoskeletal protein involved in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Ecl western blotting solution, by Bio-Rad (1 mentions)
Insulin, by Merck Group (1 mentions)
Jnk no 9252, by Cell Signaling Technology (1 mentions)
P erk thr202 tyr204, by Cell Signaling Technology (1 mentions)
Mouse insulin enzyme linked immunosorbent assay elisa kit, by Mercodia (1 mentions)
P ikkα β, by Cell Signaling Technology (1 mentions)
Antibodies against bax and bcl 2, by Santa Cruz Biotechnology (1 mentions)
Hybond c nitrocellulose membrane, by Cytiva (1 mentions)
Hrp conjugated rabbit anti mouse igg, by Agilent Technologies (1 mentions)
Swine anti rabbit igg, by Agilent Technologies (1 mentions)
Hybond, by Cytiva (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Agilent Technologies (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Santa Cruz Biotechnology (1 mentions)
Bovine serum albumin (bsa), by HiMedia (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Gel imager machine fluor chem e, by Cell Biosciences (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Compound c, by Merck Group (1 mentions)
Thiazolyl blue tetrazolium bromide, by Merck Group (1 mentions)
6 protocols using primary antibody against β actin
1
Western Blot Analysis of Signaling Proteins
2
Bovine α-Lactalbumin Enzymatic Hydrolysis
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Bovine α-lactalbumin was provided by Davisco Foods International Inc. (Eden Prairie, MN, USA). Alcalase was purchased from Novozymes Biologicals Inc. (Bagsvaerd, Denmark). Insulin was purchased from Sigma-Aldrich (St. Louis, MO, USA). The mouse Insulin enzyme-linked immunosorbent assay (ELISA) kit was obtained from Mercodia (Uppsala, Sweden). The primary antibody against β-actin (No. ab8227) was purchased from Abcam (Cambridge, UK). The primary antibodies against p-IRS-1 (Ser307) (No. 2381), p-IRS-1 (612) (No. 2386), IRS-1 (No. 3407), p-Akt (Ser473) (No. 4060), Akt (No. 4691), p-IKKα/β (No. 2597), IKKα (No. 2682), IKKβ (No. 2370), p-p38 (Thr180/Tyr182) (No. 4511), p38 (No. 8690), phospho-extracellular signal regulated kinases (p-ERK) (Thr202/Tyr204) (No. 4370), ERK (No. 4696), phospho-c-Jun N-terminal kinases (p-JNK) (Thr183/Tyr185) (No. 4668), JNK (No. 9252), and horseradish peroxidase-conjugated anti-rabbit secondary antibody (No. 7074) were obtained from Cell Signaling Technology (Beverly, MA, USA). All other chemicals used in the study were at least of analytical grade.
3
Protein Expression Analysis by Western Blot
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Cells were lysed in urea lysis buffer (7 M urea, 0.1 M DTT, 0.05% Triton X-100, 25 mM NaCl, 20 mM Hepes pH 7.5). Equal amounts of protein were separated by SDS polyacrylamide gel electrophoresis (SDS PAGE), transferred to Hybond-C nitrocellulose membrane (Amersham Pharmacia Biotech, Buckinghamshire, UK) and hybridised to an appropriate primary antibody and HRP-conjugated secondary antibody for subsequent detection by ECL. Primary antibody against β-actin was purchased from Abcam (Cambridge, UK). Antibodies against Bax and Bcl2 were purchased from Santa Cruz Biotechnology (CA, USA). The secondary antibodies, HRP-conjugated rabbit anti-mouse IgG and swine anti-rabbit IgG, were obtained from DakoCytomation (Glostrup, Denmark).
4
Quantitative Western Blot Analysis
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Whole cell lysates were obtained by resuspension of cell pellets in 500 μl RIPA buffer (Sigma, USA) and 40 μl of protease inhibitor cocktail (Sigma, USA). The lysates were estimated for protein concentration using BCA Assay method. Protein extracts (40 μg) were subjected to SDS-PAGE using 12% Tris/HCl SDS gels and transferred onto polyvinylidene difluoride (PVDF) membranes (Membrane Technologies, India). Membranes were blocked using 3% BSA (Himedia, India) for 2 hours, followed by incubation with primary antibody against β- actin (Abcam,UK, 1:500) and tyrosine hydroxylase (Santa Cruz, USA, 1:500) in 1% BSA- phosphate saline buffer (PBS) overnight at 4°C. Post incubation, membranes were washed thrice in TBST and incubated with the appropriate horseradish peroxidase (HRP) - conjugated secondary antibody (1/4,000) (Dako, USA) for 2 hours at room temperature. Membranes were developed with chemiluminescence detection reagents (Pierce, USA) and acquired by using Gel Imager machine (Fluor Chem E, Cell Biosciences, Australia).
5
Glucocorticoid Receptor Regulation Study
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GW6471 and MK 886 were purchased from Tocris Bioscience (Bristol, UK). UA, Compound C, RU486, dexamethasone, cortisol, corticosterone, and thiazolyl blue tetrazolium bromide (MTT reagent) were obtained from Sigma (St. Louis, MO). A kit for assaying lactate dehydrogenase (LDH) was purchased from Roche (Indianapolis, IN). [3H]dexamethasone was obtained from American Radiolabeled Chemicals (St. Louis, MO). Human recombinant GR was purchased from Invitrogen (Grand Island, NY). Primary antibodies against phosphorylated AMPK (Thr172) and AMPK were obtained from Cell Signaling Technology (Danvers, MA), while primary antibody against β‐actin was purchased from Abcam (Cambridge, MA).
6
Investigating Smad2 Signaling Modulation
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Cells were
seeded onto 6-well plates with FBS-free 1640 medium and cultured for
24 h. After treatment with 1, 10, and 25 μM of8 and 9 (Figure 5 ), cells were collected, and lysis buffer was added. Lysation
was allowed to proceed on ice for 30 min. Protein samples were subjected
to electrophoresis in 10% SDS–PAGE gel and transferred onto
nitrocellulose membrane (NC membrane). The membranes were blocked
in 5% BSA and incubated at 4 °C with primary antibody overnight,
followed by a secondary antibody for 1 h at room temperature. Protein
bands were detected using a LI-COR Odyssey imaging system (Lincoln,
NE, USA). Epigallocatechin gallate (EGCG) was used as the positive
control.
Primary antibodies against p-smad2
and smad2 were purchased from Cell Signaling Technology (Beverly,
MA, USA). The primary antibody against β-actin was purchased
from Abcam Inc. (Cambridge, MA, USA). Secondary antibodies, goat antimouse/antirabbit
IgG H&L (IRDye 800CW), were purchased from Abcam Inc. (Cambridge,
MA, USA).
seeded onto 6-well plates with FBS-free 1640 medium and cultured for
24 h. After treatment with 1, 10, and 25 μM of
was allowed to proceed on ice for 30 min. Protein samples were subjected
to electrophoresis in 10% SDS–PAGE gel and transferred onto
nitrocellulose membrane (NC membrane). The membranes were blocked
in 5% BSA and incubated at 4 °C with primary antibody overnight,
followed by a secondary antibody for 1 h at room temperature. Protein
bands were detected using a LI-COR Odyssey imaging system (Lincoln,
NE, USA). Epigallocatechin gallate (EGCG) was used as the positive
control.
Primary antibodies against p-smad2
and smad2 were purchased from Cell Signaling Technology (Beverly,
MA, USA). The primary antibody against β-actin was purchased
from Abcam Inc. (Cambridge, MA, USA). Secondary antibodies, goat antimouse/antirabbit
IgG H&L (IRDye 800CW), were purchased from Abcam Inc. (Cambridge,
MA, USA).
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